PRC2 disruption in cerebellar progenitors produces cerebellar hypoplasia and aberrant myoid differentiation without blocking medulloblastoma growth

We show that Polycomb Repressive Complex-2 (PRC2) components EED and EZH2 maintain neural identity in cerebellar granule neuron progenitors (CGNPs) and SHH-driven medulloblastoma, a cancer of CGNPs. Proliferating CGNPs and medulloblastoma cells inherit neural fate commitment through epigenetic mechanisms. The PRC2 is an epigenetic regulator that has been proposed as a therapeutic target in medulloblastoma. To define PRC2 function in cerebellar development and medulloblastoma, we conditionally deleted PRC2 components Eed or Ezh2 in CGNPs and analyzed medulloblastomas induced in Eed-deleted and Ezh2-deleted CGNPs by expressing SmoM2, an oncogenic allele of Smo. Eed deletion destabilized the PRC2, depleting EED and EZH2 proteins, while Ezh2 deletion did not deplete EED. Eed-deleted cerebella were hypoplastic, with reduced proliferation, increased apoptosis, and inappropriate muscle-like differentiation. Ezh2-deleted cerebella showed similar, milder phenotypes, with fewer muscle-like cells and without reduced growth. Eed-deleted and Ezh2-deleted medulloblastomas both demonstrated myoid differentiation and progressed more rapidly than PRC2-intact controls. The PRC2 thus maintains neural commitment in CGNPs and medulloblastoma, but is not required for SHH medulloblastoma progression. Our data define a role for the PRC2 in preventing inappropriate, non-neural fates during postnatal neurogenesis, and caution that targeting the PRC2 in SHH medulloblastoma may not produce durable therapeutic effects. Supplementary Information The online version contains supplementary material available at 10.1186/s40478-023-01508-x.


Introduction
During brain development, epigenetic inheritance specifies cell identities, directing progenitor cell differentiation along trajectories determined by their lineage. Thus, rhombic lip progenitors give rise to progeny with specific neural fates, including cerebellar granule neurons (CGNs) [1] and unipolar brush cell neurons (UBCs) [2]. Lineage tracing by scRNA-seq shows that CGNPs differentiate into neurons and not into other types of cells [3]. However, hyperactivation of SHH signaling can transform CGNPs, resulting in medulloblastoma [4-7], a primitive neuro-ectodermal tumor that is the most common malignant pediatric brain tumor. While most medulloblastoma cells adhere to a neural fate trajectory [8,9], a small fraction of tumor cells differentiates as glia [3,10]. In giving rise to glia, SHH medulloblastoma cells demonstrate increased pluripotency beyond the neural fate trajectory of CGNPs but maintain commitment to typically neuro-ectodermal fates. Epigenetic mechanisms thus maintain neuroectodermal lineage commitment through generations of proliferating progenitors during brain development and through generations of proliferating tumor cells in medulloblastoma.
PRC2 is a chromatin regulatory complex that has been shown to positively or negatively regulate neural progenitor proliferation in different contexts. The core subunits of the PRC2 include EZH1/2, EED, SUZ12, and RbAp46/48. By regulating trimethylation of H3K27 (H3K27me3), PRC2 suppresses CDKN2A, thus increasing neural progenitor proliferation in the hippocampus [11]. However, PRC2 also inhibits SHH-induced transcriptional regulation by depositing H3K27me3 at bivalent chromatin sites in the promoter regions of SHH pathway target genes [12]. As SHH signaling drives CGNP proliferation [13,14] and medulloblastoma tumorigenesis [7, [15][16][17][18], PRC2-mediated repression of SHH target genes suggests the potential to inhibit proliferation in SHH-driven cells. In addition to affecting proliferation, PRC2 regulates hippocampal progenitor differentiation [11], consistent with of its role in fate commitment in diverse developmental contexts, from drosophila larva [19] to mammalian embryonic stem cells [20,21]. PRC2 may similarly contribute to cerebellar development by regulating CGNP proliferation and differentiation.
As in brain development, prior studies have shown divergent roles for the PRC2 in cancer, positively or negatively regulating tumor growth in different types of tumors [64,65]. SHH medulloblastomas upregulate EZH2, the catalytic subunit of the PRC2, suggesting a growth-promoting function [12], and EZH2 inhibition has been proposed as a medulloblastoma therapy [22]. A more complex relationship, however, is suggested by the finding that pharmacologically increasing H3K27me3 levels in cultured medulloblastoma cells decreases tumor cell viability [12]. Moreover, prior studies show different effects on tumor growth in murine medulloblastoma models with widespread or mosaic Eed deletion [23]. The role of EZH2 in catalyzing inhibitory H3K27me3 marks and its non-canonical PRC2-independent functions has raised questions about the therapeutic potential of EZH2 inhibition [24][25][26].
To determine how PRC2 components affect cerebellar development, we deleted Eed conditionally in the CGNP-specific Atoh1. We then compared CGNP proliferation, apoptosis, differentiation, and gene expression in the resulting Eed-deleted mice, Eed-intact controls, and to mice with Ezh2-deleted CGNPs. To determine PRC2 function in SHH medulloblastoma, we bred Eed-deleted and Ezh2-deleted mouse lines with mice genetically engineered to develop SHH medulloblastoma from CGNPs. Our data show that CGNPs and medulloblastoma cells require PRC2 to maintain neural fate commitment, and that EED is specifically required for cerebellar growth, but neither EED nor EZH2 are required for medulloblastoma progression.

EED is required for proper cerebellar growth
CGNPs showed robust EED and EZH2 expression during postnatal neurogenesis, with initially low but detectable H3K27 trimethylation that increased as CGNPs differentiated (Fig. 1A). To reduce variation in developmental age when making comparison between genotypes, we used Eed f/f littermates of Eed cKO mice to represent normal development. These mice did not inherit Cre and showed intact EED expression. During the period of CGNP proliferation from postnatal day 1 (P1) through P15, cells of the CGNP lineage segregate spatially according to developmental state, with undifferentiated and early differentiating CGNPs in the external granule cell layer (EGL) and differentiated, post-mitotic CGNs in the internal granule cell layer (IGL). At P7, CGNPs throughout the EGL expressed EED and EZH2, and H3K27me3 was detectable in the EGL and IGL (Fig. 1A). H3K27me3 increased in the CGNs of the IGL by P21. Expression of PRC2 components thus continued throughout CGNP development and H3K27me3 correlated with neuronal maturation, suggesting a role for the PRC2 in the differentiation process.
In light these data, we analyzed Eed and Ezh2 function in CGNP using conditional genetic deletion. Prior studies showed that conditional Ezh2 deletion in the dorsal neural tube at embryonic day E12.5 results in cerebellar hypoplasia, with decreased numbers of both Purkinje cells and CGNPs [27]. However, the loss of Purkinje neurons may indirectly alter CGNPs, which depend on SHH released by Purkinje cells in order to proliferate [28]. To disrupt PRC2 genes specifically in CGNPs, we used conditional deletion by expressing Cre from the Atoh1 (aka Math1) promoter, which in the cerebellum is CGNP specific [29]. We interbred Math1-Cre transgenic mice that express Cre recombinase in the CGNP population with mice harboring conditional alleles of either Eed (Eed fl/fl ) or Ezh2 (Ezh2 fl/fl ), to generate Math1-Cre/Eed f/f (Eed cKO ) and Math1-Cre/Ezh2 f/f (Ezh2 cKO ) mice. Eed deletion resulted in marked depletion of the CGNP lineage, with markedly less populous EGL in 5/5 P7 Eed-cKO cerebella compared to controls (Fig. 1B). All Eed cKO mice subjected to immunohistochemistry (n = 3) showed loss of both EED protein and EZH2 protein throughout the EGL (Fig. 1B), consistent with reports that EED loss destabilizes EZH2 [30]. H3K27me3 expression was absent in the Eed cKO CGNPs, consistent with PRC2 disruption (Fig. 1B). In contrast, Purkinje cells showed robust EED, EZH2, and H3K27me3; however, these cells, which typically localize at the outer margin of the CGNs, were scattered throughout the depopulated IGL and remained identifiable by their typical morphology with large nuclei and cell bodies (Fig. 1B, red arrows). At P21, 5/5 Eed cKO cerebella were clearly hypoplastic, with no clear IGL, and with inappropriately persistent, small populations of H3K27me3 + cells in the EGL ( Fig. 1B; white arrow). Consistent with cerebellar impairment, Eed cKO mice showed tremor and ataxia and frequently fell while walking. EED was thus required for PRC2 stability, demonstrated by loss of EZH2 protein in Eed cKO CGNPs, for H3K27 tri-methylation in CGNPs, and for cerebellar growth and function.
Ezh2 deletion in contrast did not cause neurologic abnormalities or overt cerebellar hypoplasia (Fig. 1C). P7 Ezh2 cKO CGNPs showed intact EED protein expression despite widespread absence of EZH2 (Fig. 1C). H3K27me3 was clearly reduced in Eed cKO CGNPs and CGNs compared to controls (Fig. 1C). Thus, while H3K27me3 was altered in both Eed cKO and Ezh2 cKO mice, Eed deletion more effectively disrupted the PRC2 complex, as demonstrated by loss of EZH2 in Eed cKO cerebella, and more profoundly altered cerebellar growth.
We compared chromatin marks in Eed cKO and Ezh2 cKO cerebellar lysates, using controls mice that did not inherit the Cre transgene. Western blot analysis showed that both Eed and Ezh2 deletions disrupted PRC2 function and we did not identify differences in chromatin marks. Both deletions reduced H3K27me3 as expected (Fig. 1D). However, residual H3K27me3 from cerebellar cells outside the Atoh1 lineage that were not subject to conditional deletion produce detectable signal that may have obscured differences. We noted a trend toward increased H3K27 acetylation in both Eed cKO and Ezh2 cKO (Fig. 1D) that was not statistically significant but would be consistent with the previously observed increased H3K27Ac in H3K27me3-depleted embryonic stem cells [31,32]. Eed cKO CGNPs did not show differences in H2AK119 monoubiquitination, indicating that PRC1 function was not altered, and did not show differences in H3K4me3 (Fig. 1E). Within the resolution of our western blot assays, therefore, deletion of Eed or Ezh2 resulted in similar chromatin changes, limited to H3K27 modification.

Eed deletion decreases progenitor proliferation and increases apoptosis
We investigated developmental processes that mediate growth failure in Eed cKO cerebella. Eed deletion was previously shown to decrease proliferation and increased apoptosis in hippocampal progenitors [11]. As both decreased CGNP proliferation and increased CGNP apoptosis can cause cerebellar hypoplasia [13,[33][34][35], we quantified proliferative CGNPs, identified by expression of phosphorylated-RB (pRB) and apoptotic CGNPs, identified by expression of cleaved Caspase-3 (cC3). We used as controls littermates of Eed cKO mice that had at least one intact Eed allele, including Eed f/f mice and Math1-Cre/Eedf /+ mice. Similar to hippocampal progenitors, Eed cKO CGNPs showed decreased proliferation and increased apoptosis (Fig. 1F), implicating both processes in the cerebellar hypoplasia of Eed cKO mice.

Eed deletion produces discrete patterns of transcriptomic change
To resolve the effects of Eed-deletion in individual cells, we subjected Eed-deleted and control CGNPs to scRNAseq analysis. We harvested cerebella from 3 replicate P7 Eed cKO mice and subjected them to Drop-seq bead-based scRNA-seq preparation [66]. We then sequenced the resulting bar-coded libraries and identified cells by beadspecific bar codes. After QC and filtering, we included 2377 cells for analysis. We compared these cells to previously sequenced data from 6631 cells from 5 replicate P7 WT cerebella. To adjust for differences in sequencing depth in Eed cKO and WT cells, we down-sampled the WT cells to 46.5% of their original depth to achieve similar sequencing depth between conditions, consistent with best practices [36].
We subjected scRNA-seq data from Eed cKO and WT cells to principal component analysis (PCA) and Louvain clustering, as in our prior studies [3, 37,38], to identify 20 clusters with distinctive gene expression, numbered from 0, most populous, to 19, least populous (Additional file 1: Fig. S1). We then determined cluster-specific gene expression profiles by comparing the expression of each detected gene in cells within the cluster versus all cells outside the cluster (Additional file 2: Data 1). In Clusters 4 and 6, we noted that cluster-specific genes were expressed by discrete subpopulations, suggesting that further sub-clustering of these clusters would be informative. Re-iterative clustering split Cluster 4 into 4_0 and 4_1 and Cluster 6 into 6_0 and 6_1, which showed discrete, cluster-specific patterns of gene expression (Additional file 2: Data 1). We mapped these 22 clusters by color code on the UMAP projection to visualize the different populations ( Fig. 2A).
The differential gene expression patterns identified the cell-type of each cluster and demonstrated diverse types of cells that were expected in cerebellar tissue ( Fig. 2A,B; Table 1). We identified Clusters 0, 1, 2, 3, 4_1, 5, 6_1, 10, 14, 16, and 17 as CGNP lineage cells in a spectrum of differentiation states, from cycling CGNPs to differentiated CGNs, based on expression of SHH target genes including Sfrp1, neural progenitor-related genes including CGNP transcription factor Barhl1 and axonal pathfinding receptor Cntn2, and neuronal genes including the transcription factor Rbfox3. Different markers identified diverse types of non-neural cells, including astrocytes (Cluster 7), oligodendrocytes (Cluster 9), vascular fibroblasts (Cluster 11), myeloid cells (Cluster 12), endothelial cells (Cluster 13), pericytes (Cluster 18), and ependymal cells (Cluster 19). Markers also identified neural populations that were outside the Atoh lineage but expected in the cerebellum, including gabaergic neural progenitors (Cluster 6_0), identified by expression of Ascl1 and Pax3, gabaergic interneurons (Cluster 4_0) identified by Pax2 and Gad1, and Purkinje cells identified by Gad1 and Calb1 [3,39]. In contrast to all other clusters, which demonstrated markers expected in the brain, Cluster 15 expressed the muscle cell marker Myog, suggesting myoid differentiation.
We disaggregated the UMAP to make separate projections from Eed cKO and WT mice, demonstrating that each genotype distributed differently across the clusters (Fig. 2C). To compare the cluster populations in Eed cKO and WT samples statistically, since each replicate contributed different numbers of cells to the analysis, we normalized the population of cells in each cluster from each replicate to the total number of cells from that replicate. These proportional populations were inter-related by the normalization to the whole, and therefore could not be analyzed by individual t-tests, which assume independence. We therefore used Dirichelet regression analysis to compare the proportional cluster populations [40].
As suggested by the disaggregated UMAP (Fig. 2C), Dirichelet regression analysis showed that the populations of specific clusters were significantly different in Eed cKO cerebella compared to controls (Fig. 2D). We found significant differences in the populations of Atoh1-lineage cell types that were subject to conditional deletion, and also of clusters outside the Atoh1 lineage. Within the Atoh1 lineage, CGNP/CGN Clusters 0, 1, 2, 3, 10, and 16 were more populous in controls, while CGNP/ CGN Clusters 4_1, 5, 6_1, and 8 were more populous in the Eed cKO (Fig. 2D). Outside the Atoh1 lineage, Purkinje cells, astrocytes, and vascular fibroblasts were increased in controls and pericytes were increased in the Eed cKO . These cell types were not subject to Eed deletion, and differences in their populations reflect non-cell autonomous effects. Additionally, the myoid Cluster 15 was present only in the Eed cKO cerebella (Fig. 2C,D). While proliferative Clusters 5 and 6_1 were relatively increased in Eed cKO mice, proliferative Clusters 0, 3, and 10 were decreased, resulting in a net decrease in proliferative populations (Fig. 2E), consistent with the decrease in pRB + CGNPs.

Eed deletion permits divergence from the neural fate of CGNPs
The Myog-expressing Cluster 15 showed multiple genes typical of muscle cells, including troponins, Myl1, Cav3, and Smyd1 (Fig. 3A). We did not observe this myoid transcriptomic pattern in any cells from control mice. Cluster 15 cells also up-regulated Cdkn1a and Cdkn1c (Fig. 3A,B), which are known to be suppressed by the PRC2 in other types of cells [41][42][43][44]. Based on the upregulation of PRC2-suppressed genes, we infer that Cluster 15 cells were within the Atoh1 lineage, and that conditional Eed deletion caused these Atoh1-lineage cells to diverge from the expected CGNP trajectory.  Hoxa7 in WT cells (Fig. 3B) indicates that each of these genes are normally suppressed by the PRC2 in CGNPlineage cells.

Ezh2 deletion, like Eed deletion, permits myoid differentiation
We used immunohistochemistry (IHC) to determine if specific transcripts from PRC2-target genes were translated in Eed cKO cerebella, and to probe Ezh2 cKO cerebella for similar patterns of expression. Cells expressing CDKN2A, MYOG, and SMYD1 were absent in controls, including WT cerebella and cerebella from Eed f/f and Eed f/+ littermates of Eed cKO mice. As the frequency of CDKN2A + , MYOG + , and SMYD1 + cells in mutant cerebella could not be lower than in control cerebella, we used one-tailed statistical tests to compare mutant to control replicates, and two-tailed statistical tests for comparison between Eed cKO and Ezh2 cKO genotypes. Eed cKO cerebella showed CDKN2A + CGNPs throughout the EGL; the fraction of CDKN2A + cells was significantly greater than in WT or Ezh2 cKO cerebella (Fig. 3C). Ezh2 cKO cerebella showed significantly more CDKN2A + CGNPs than controls, but fewer than Eed cKO cerebella. Both PRC2 component mutations thus resulted in up-regulation of CDKN2A, with Eed cKO showing a higher fraction of affected cells.
Both Eed ckO and Ezh2 ckO cerebella showed cells expressing the muscle cell transcription factor MYOG, while no cells expressed MYOG control cerebella, Eed cKO cerebella (Fig. 3D). MYOG + cells were significantly more numerous in Eed ckO than Ezh2 cKO cerebella (Fig. 3D). Eed ckO cerebella also showed cells expressing the muscle cell chromatin regulator SMYD1, which was absent in Ezh-2 ckO and control cerebella (Fig. 3D). Pairwise comparison of SMYD1 expression in Eed ckO cerebella (present in 3/3) versus Ezh2 ckO (absent in 3/3) or controls (absent in 3/3) using the Barnard's exact test showed p values of 0.03. Transcripts that were not found in control CGNPs, including Cdkn2a and Myog, were thus translated into proteins in both Eed ckO and Ezh2 ckO cerebella, with SMYD1 expression in Eed cKO cerebella demonstrating an additional myoid gene ectopically expressed.

Increased activation of the intrinsic apoptotic pathway in Eed cKO CGNPs
To probe the mechanisms of increased cell death in Eed-cKO CGNPs, we analyzed p53 function and the expression  of genes regulating apoptosis. CGNPs are highly sensitive to p53-mediated activation of the intrinsic apoptotic pathway [48,49] and also to direct activation the intrinsic apoptotic pathway triggered by changes in apoptotic regulators [35,50]. Cdkn1a expression in Cluster 15 suggested that Eed deletion might activate p53-dependent transcription, potentially mediating the observed increased apoptosis. To determine if other apoptotic regulators were altered in Eed cKO CGNPs, we compared the expression of pro-apoptotic BH3-only genes in the CGNP and CGN clusters of Eed cKO and control mice (Fig. 4A).
Hierarchical clustering based on BH3-only gene expression distinguished the 3 proliferative clusters enriched in Eed cKO cerebella, Clusters 4_1, 5, and 6_1, which showed increased expression of Pmaip1 (aka Noxa), Bad, Bcl2l11 (aka Bim), and Hrk (Fig. 4A). Similarly, comparing the combined set of all cells in CGNP and CGN clusters, Eed cKO cells showed a distinctive pattern of BH3-only gene expression, with increased Bbc3 (aka Puma), Pmaip1 (aka Noxa), Bad, Bcl2l11 (aka Bim), and Hrk (Fig. 4B). These data suggested that Eed deletion may increase apoptosis by direct activation of the intrinsic apoptotic pathway.
To determine whether p53 signaling or the intrinsic apoptotic pathway contributed to cerebellar hypoplasia in Eed cKO mice, we combined Eed deletion with deletion of either Trp53 or both Bax and Bak. We bred  (Fig. 4C). In contrast, cerebella in Eed/Bax/Bak tKO mice were markedly less abnormal, with relatively increased CGNs within the IGL and more appropriate layering of Purkinje cells between the IGL and molecular layers (Fig. 4C). Bax/Bak co-deletion did not fully rescue the effects of Eed knockout, as the IGL remained less densely populated than WT controls and the molecular layer contained ectopic cells (Fig. 4C). The absence of rescue in Eed/Trp53 dKO mice indicates that growth failure in the Eed cKO cerebella was p53-independent. The partial rescue by co-deletion of Bax and Bak, however, demonstrates that p53-independent activation of the intrinsic apoptosis pathway contributed to cerebellar hypoplasia in Eed-cKO mice. Bax/Bak co-deletion also increased the myoid population (Additional file 1: Fig. S2), indicating that myoid cells were typically removed from Eed cKO cerebella by apoptosis.

PRC2 function is not required for SHH medulloblastoma tumorigenesis
Mutations that disrupt cerebellar growth may identify genes required for growth of medulloblastoma [51] and the PRC2 has been proposed as a target for medulloblastoma therapy. Therefore, to determine whether medulloblastomas depend on Eed and the PRC2, we bred Eed cKO and Ezh2 cKO mice with SmoM2 mice [52].  (Fig. 5E). PRC2 disruption therefore decreased both apoptosis and terminal differentiation in SHH medulloblastoma.  (Fig. 5A), suggesting that Eed deletion might produce an initial growth suppression, followed by more rapid growth.
To analyze tumor growth dynamics, we compared RB  (Fig. 5J). The smaller pRB + population in M-Smo/Eed cKO tumors at P12, the increased rate of proliferation within the pRB + population, and the similar pRB + population at P18 are all consistent with a biphasic effect of Eed deletion on tumor growth, in which tumors initially grew more slowly and then accelerated, producing shorter survival times.

Up-regulation of PRC2 target genes without growth suppression in PRC2-mutant medulloblastomas
We investigated whether medulloblastomas with deletion of Eed or Ezh2 up-regulated the same PRC2 targets that were up-regulated in  (Fig. 6D), in replicate sections, while blinded to the genotype. The resulting quantifications of myoid cells correctly distinguished control tumors from tumors with either Eed or Ezh2 deletion, which were not significantly different from each other (Fig. 6E). Deletion of either Eed or Ezh2 was therefore sufficient to allow myoid differentiation, reproducing the molecular and histologic features of medullomyoblastoma.

Discussion
Our data show that the PRC2 maintains neuronal fate commitment in cerebellar progenitors and in SHH medulloblastoma by preventing alternative, myoid differentiation. In the postnatal cerebellum, conditional deletion of PRC2 components Ezh2 and Eed in the Atoh1 lineage disrupted PRC2-mediated H3K27 trimethylation and caused a fraction of CGNPs to differentiate along a muscle cell trajectory. Eed deletion induced myoid differentiation in more cells than Ezh2 deletion and markedly impaired cerebellar growth through a combination of decreased proliferation and increased apoptosis. In medulloblastomas, deletion of either Eed or Ezh2 resulted in myoid differentiation, but neither deletion increased apoptosis or durably prevented tumor growth. Rather, deletion of either Eed or Ezh2 accelerated tumor progression. Single-cell transcriptomic analysis of postnatal cerebella showed that Eed-deleted CGNPs inappropriately expressed genes typically suppressed by the PRC2, including Cdkn2a, Hoxa9, and Hoxa7. Up-regulation of Cdkn2a may be sufficient to explain reduced CGNP proliferation, as seen in Eed-deleted hippocampal progenitors [11]. Up-regulation of specific BH3-only genes suggested that direct activation of BAX or BAK may mediate increased CGNP apoptosis. Co-deletion studies of Eed plus either Trp53 or Bax AND Bak confirmed that increased apoptosis occurred by p53-independent activation of the intrinsic apoptosis pathway. The partial rescue of cerebellar growth in Eed/Bax/Bak tKO mice demonstrates that inappropriate apoptosis contributed to growth failure and restricted the myoid population.
PRC2 function was not required for neural differentiation, as in Eed-deleted cerebella, most CGNPs were able to complete neural differentiation, achieving the neural fates of Clusters 4_1 and 8 in Eed cKO mice. The inappropriate expression of Hox genes in these clusters did not prevent a recognizable CGN-like pattern of gene expression. In contrast, the myoid differentiation of Cluster 15 demonstrates that PRC2 disruption permitted new fate possibilities.
The specific diversion of CGNPs and medulloblastoma cells into myoid fates may be related to the normal expression of the MYOD1 transcription factor during postnatal development. MYOD1 is an early myogenic transcription factor that activates MYOG in developing muscle progenitors. Prior studies show that WT P7 cerebella and SHH medulloblastomas contain populations of cells that express MYOD1 without inducing MYOG or activating a myogenic program [3,54]. In the Eed-deleted and Ezh2-deleted cerebella and medulloblastomas, however, Myod1+ cells also expressed Myog and adopted a myogenic trajectory. These data suggest that one function of the PRC2 in CGNPs is to suppress Myog and other myoid genes, allowing CGNPs to use the MYOD1 transcription factor to regulate neural development, without risk of inappropriate differentiation. We propose more generally that by suppressing inappropriate differentiation pathways, the PRC2 allows specifically neural functions of transcriptional regulators such as MYOD1, that have non-neural functions in other types of cells.
Eed deletion resulted in cerebellar hypoplasia that was not seen in Ezh2-deleted mice. The difference in phenotype may result from a more severe PRC2 disruption caused by loss of EED. In other cell types, EED protein is required for the stability of the other components of the PRC2 [30] and for PRC2 methyltransferase activity [55]. Consistent with these prior reports, we found that Eed-cKO CGNPs lacked both EED and EZH2 proteins, indicating destabilization of the entire PRC2. In contrast, EZH2 is not required for PRC2 stability and can be partially compensated by the homolog EZH1 in multiple cellular contexts [56,57]. The PRC2 components that persist in Ezh2-deleted CGNPs, possibly with compensation from EZH1, may retain sufficient function to sustain cerebellar growth, and to suppress PRC2 target gene expression in most but not all CGNPs, resulting in fewer myoid cells in Ezh2 cKO cerebella compared to Eed cKO cerebella. Different phenotypes were similarly noted when either Eed or Ezh2 were deleted in intestinal epithelia [44]. Conditional deletion of Eed in the intestinal crypts decreased proliferation and caused hypoplasia, while Ezh2 deletion did not cause an overt phenotype. The continued proliferation in Ezh2-deleted intestinal crypts suggests that EZH1 may compensate for EZH2 loss in these cells [44], and a similar mechanism may explain sustained proliferation of Ezh2-deleted CGNPs.
Alternatively, as a shared component of the PRC1 and PRC2 complexes, EED loss may more broadly affect chromatin repression [58]. Our finding that Eed deletion did not affect levels of H2AK119 monoubiquitylation suggests that PRC1 activity was not altered in Eed cKO CGNPs or medulloblastomas. The overlapping patterns of differential gene expression in Eed cKO and Ezh2 cKO cerebella show that suppression of myoid differentiation in CGNPs depends on PRC2 function. We cannot, however, exclude the possibility that the growth-suppressive effects of Eed deletion are mediated by functions of EED protein outside of the PRC2.
In SHH medulloblastomas, disrupting PRC2 activity through deletion of either Eed or Ezh2 was sufficient to allow widespread expression of genes typically suppressed by the PRC2, including the CDKN2A tumor suppressor. Neither PRC2 disruption nor CDKN2A expression, however, was sufficient for sustained suppression of tumor growth. Eed deletion reduced the overall proliferation rate in each P12 M-Smo/Eed cKO tumor, producing transient growth suppression. Over time, however, a fraction of Eed-deleted tumor cells that were rapidly proliferative increased, driving ultimately faster progression. The initial reduction in tumor growth in M-Smo/Eed cKO tumors was consistent with previous studies that showed anti-tumor effects of PRC2 disruption using EZH2 inhibitor treatment in models SHH medulloblastoma [22,59,60]. However, the shorter survival times in M-Smo/Eed cKO and M-Smo/Ezh2 cKO mice raise concern that PRC2 disruption may not produce durable anti-tumor effects.
Our genetic studies identify a role of PRC2 in regulating neural fate commitment of cerebellar progenitors and medulloblastoma cells, and implicate PRC2 disruption in the pathogenesis of medullomyoblastoma, a subtype of medulloblastomas characterized by myogenic differentiation of tumor cells [61,62]. Our data also caution that pharmacologically inhibiting PRC2 function in medulloblastoma may hasten, rather than slow, tumor growth.

Mice
We generated Ezh2 cKO mice by breeding All mice were of species Mus musculus and crossed into the C57BL/6 background through at least five generations.

Histology and immunohistochemistry
Mouse brains were processed, immunostained, and quantified as previously described [67][68][69]. In brief, mice were placed under isoflurane anesthesia and decapitated. Harvested brains were fixed by immersion in 4% formaldehyde for 24 h and then transferred to a graded ethanol series and embedded in paraffin and sectioned along the sagittal midline. Samples were stained and imaged using an Aperio Scanscope and quantified via automated cell counting using Tissue Studio (Definiens).

Flow cytometry: cell cycle analysis
P12 and P18 M-Smo and M-Smo/Eed cKO mice were injected with Edu 1 h prior to harvest. Mice were anesthetized with isoflurane and decapitated. Tumors samples were dissociated using the Cell Dissociation Kit (Worthington Biochemical Corporation, #LK003150), which included dissociation with papain at 37 °C for 15 min, and isolation using an ovomucoid inhibitor density gradient. Tumor cells were then treated with the Fixation and Permeabilization Kit (Life Technologies, #GAS004), and stained using the following antibodies: 647-conjugated pRB diluted 1:50 (Cell Signaling, #8974) and FxCycle Violet at 1:100 (Life Technologies, #F10347). Edu was detected using a Click-iT EdU Alexa Fluor 488 Imaging Kit (catalogue number C10337; Life Sciences). Samples were run on an LSRFortessa (BD Biosciences) at the UNC Flow Cytometry Core. Data was analyzed using Flow Jo v10.

Single cell sequencing (scRNA-seq): sample collection
Brains were harvested and cut along the sagittal midline. One half of the cerebellum from each mouse was dissociated and processed for scRNA-seq, and the other half of the brain was fixed, sectioned, and analyzed to confirm phenotype. Half cerebella were processed using the Cell Dissociation Kit (Worthington Biochemical Corporation, #LK003150), in which samples were treated with papain at 37 °C for 15 min and then separated by centrifugation of an ovomucoid inhibitor density gradient. Cells were then subjected to bead pairing by microfluidics, cDNA synthesis, and library construction using the Drop-seq V3 method [66] as in our prior studies [70][71][72][73].

scRNA-seq: processing data
Data analysis was performed using the Seurat R package version 3.1.1 [74]. Data were subjected to several filtering steps. Genes detected in > 30 cells were filtered out, to prevent misaligned reads appearing as rare transcripts in the data. Putative cells with fewer than 500 detected RNA molecules (nCount) or 200 different genes (nFeature) were considered to have too little information to be useful, and potentially to contain mostly ambient mRNA reads. Putative cells with greater than 4 standard deviations above the median nCount or nFeature were suspected to be doublets, improperly merged barcodes, or sequencing artifacts, and were excluded. As in our previously published work, putative cells with more than 10% mitochondrial transcripts were suspected to be dying cells and also excluded [70].
In total, 86% of putative cells from WT mice and 74% of putative cells from Eed cKO mice met QC criteria and were included in the analysis. From the 5 WT mice, we included a total of 6558 cells with a range of 673-1852 cells per animal and a median of 1138 cells. From the 3 Eed cKO mice, we included a total of 2576 cells, with a range of 692-1036 cells per animal and a median of 847 cells.
scRNA-seq: data normalization, clustering, differential gene expression, and cell type identification The data was normalized using the SCTransform method as implemented in Seurat. The function then selected the top 3000 most highly variable genes. PCA was performed on the subset of highly variable genes using the RunPCA function. We used 15 PCs in downstream analysis, based on examining the elbow in the elbow plot as implemented by Seurat. We identified cell clusters using the FindNeighbors and FindClusters functions.
To identify differential genes between clusters of cells, we used the Wilcoxon rank sum test to compare gene expression of cells within the cluster of interest to all cells outside that cluster, implemented by the FindMarkers function. Uniform Manifold Approximation and Projection (UMAP) was used to reduce the PCs to two dimensions for data visualization using the RunUMAP function. For re-iterated analysis of the Clusters 4 and 6, the same procedures were used. We then determined the type of cell within each cluster by analyzing cluster-specific gene expression patterns.

Survival curves
Tumor-bearing mice were monitored daily and harvested according to a pre-determined humane endpoint, which included a decrease of weight > 10% overnight, a hunched posture, decreased mobility or inability to eat, and ataxia.

Statistical analyses
Two-tailed Student's t-tests were used to compare IHC and western blot quantifications between Ezh2 cKO and Eed cKO genotypes. One-tailed Student's t-tests were used to compare these genotypes versus controls for markers that were absent in controls. The Barnard's exact test was used to make comparisons between categorical variables in comparisons of markers that were determined to be present or absent in individual replicates. Survival curves were compared using the Logrank (Mantel-Cox) test. Dirichelet regression analysis was performed in R using the DirchletReg 0.7-1 package [63].
Additional file 1: Figure S1. Initial uniform manifold approximation and projection (UMAP) qualitative map of cells dissociated from harvested cerebella from 5 WT and 3 Eed cKO mice. Cells were subdivided into 20 color-coded clusters. Figure S2 Disabling apoptosis increased the myoid population. Representative images of MYOG IHC in cerebella of indicated genotypes show that a population of myoid cells persisted in Eed cKO cerebella at P21, and that the myoid population increased when apoptosis was blocked by deletion of both Bax AND Bak. The increased myoid cells in Eed/Bax/Bak tKO cerebella indicates that apoptosis decreases the myoid population in Eed cKO cerebella Additional file 2. Cluster-specific sets of differentially expressed genes, with statistical analyses.